Ca restores redox homeostasis and stress response
As shown in Table 1, a plenty of proteins involved in redox homeostasis
and stress response changed their expression abundances at various Ca
levels. All the identified antioxidant enzymes regulating redox
homeostasis showed increased or equal expression abundance at low Ca
level compared with the control, including APX (spot 52), cytosolic APX
(spot 57), chloroplast stromal APX (spot 58), tau class glutathione
S-transferase (GST, spot 59), members of SOD (spot 60-62) and 2-Cys
peroxiredoxin BAS1 (spot 66). The higher expression of antioxidant
enzymes in plants induced by various abiotic/biotic stresses has been
widely reviewed; the up-regulated enzymes enable plant to strengthen
stress tolerance and resistance through maintaining redox
homeostasis.54,55Ca deficiency is a kind of nutrient deficiency stress, and our study
hints Ca deficiency may share some common oxidative damage patterns with
other abiotic/biotic stresses that cause plant growth inhibition.
However, the total APX activity and the total SOD activity showed
declined trend at the low Ca level (Figure 1H and I), which looks
contradictory with the increased APX and SOD expression. It can be
interpreted as follow. Under long time (two months) Ca deficiency stress
in our study, the translation of antioxidant enzymes were activated and
contributed to accumulated enzymes. Nonetheless, the overall ability of
these enzymes to respond oxidative stress may decline gradually with the
elongation of stress time, these enzymes showed decreased activity and
even deactivated completely as a result of long time Ca deficiency
stress.
In the high Ca level, the majority of these antioxidant enzymes showed
declined expression. The rationalization to this phenomenon is that
adequate exogenous Ca supply relived Ca deficiency stress and subsequent
oxidative stress, therefore there is no necessity to sustain high level
of antioxidant enzymes.
What’s interesting is that cytosolic APX (spot 57), chloroplast stromal
APX (spot 58) and Mn-SOD (spot 62) showed increased abundance at both
low Ca level and high Ca level compared with the control. Low Ca
treatment induced higher APX and SOD expression may probably due to the
Ca deficiency resulted oxidative stress. While the up-regulated
cytosolic APX, chloroplast stromal APX and Mn-SOD at high Ca level
probably be related to the exogenous Ca improved P. massonianaseedling photosynthesis (Figure 1B and C). In the process of
photosynthetic electron transfer and enzymatic reaction, it is
inevitable to produce some by-products such as reactive oxygen species
(ROS) including superoxide anion radical (O2ˉ) and hydrogen peroxide
(H2O2), while
H2O2 production even acts as an index
for photosynthetic electron transport activity
evaluation.56,57Strengthened photosynthesis signifies accelerated ROS production and
accumulation.57 Within
plant cell, especially in chloroplast, O2ˉ is catalyzed to
H2O2 by SOD, and then
H2O2 is reduced to water by APX.46,47SOD and APX play critical roles in photosynthesis by regulating ROS
level.57,58High Ca treatment induced the up-regulation of cytosolic APX,
chloroplast stromal APX and Mn-SOD manifests exogenous Ca activated the
APX and SOD to remove excessive ROS, especially O2ˉ and
H2O2, and then improved photosynthesis.
The interaction between APX and SOD in the PPI network (Figure 5)
further proved their collaborative role in ROS elimination. The
increased H2O2 content, total APX
activity and total SOD activity (Figure 1G-I) under high Ca treatment
evidenced this deduction at physiological level.
Three stress response proteins, abscisic stress ripening protein 2 (spot
65), transcription factor bHLH145 (spot 48) and lipoxygenase (spot 5)
showed similar changing patterns in expression abundances with that in
antioxidant enzymes (Table 1). Transcription factor bHLH145 execute a
positive regulation of stress response, such as drought and salinity
stress response.59Lipoxygenase initiate the hydroperoxidation process of polyunsaturated
fatty acids and resulted the formation of a variety of oxylipins, such
as plant hormone jasmonic acid60. Various stresses
could activate the expression of lipoxygenase and motivate the
sequential stress response
pathways.60 Abscisic
stress ripening protein 2 showed increased abundance under drought
stress.61 Compared with
low Ca treatment, the down-regulation of the three proteins at high Ca
level further proved adequate exogenous Ca can alleviate ROS stress and
confer P. massoniana seedlings advanced environment adaptability.